ABSTRACT The human immunodeficiency virus (HIV) epidemic affects 1% of the global population, leading to dramatic economic consequences. The survival of HIV is contingent upon interactions between host and virus proteins and HIV's ability to repurpose the host's biological processes. HIV replication is dependent on the nuclear export of unspliced and singly spliced viral mRNAs; while the host, by contrast, retains unspliced RNA in the nucleus. In order to circumvent detainment of HIV transcripts, the virus utilizes a regulatory protein (Rev) and a structured region of the viral RNA to form a ribonucleoprotein (RNP) complex. Rev is generated from the fully spliced viral transcript and oligomerizes on the Rev response element (RRE), a multi-hairpin structure within the intron of unspliced and singly sliced viral transcripts. The Rev/RRE RNP hijacks Crm1, a host nuclear export receptor that is regulated by the GTPase Ran, facilitating export of the viral RNA to the cytoplasm. The structural organization of the complex remains elusive and without structures of the HIV RNP (Rev/RRE) and the nuclear export complex (Rev/RRE-hCRM1/RanGTP) it is difficult to discern the mechanism of complex formation and nuclear export. Recent biochemical and structural experiments revealed a novel Crm1 dimer that is dependent on a species-specific interface that is critical to viral replication and a flexible Rev dimer interface that undergoes large conformational changes in the presence of viral RNA. These interfaces have potential to be therapeutic targets, and details of the structure will be critical for exploring those possibilities. In this study we are interested in elucidating the overall architecture of both the HIV Rev/RRE RNP and the HIV nuclear export complex. Specifically, we want to provide explanations for the role of the Rev oligomer and Crm1 dimer, and elucidate the details of the protein-protein and protein-RNA interfaces. In aim 1 single particle cryo-electron microscopy (EM) will be used to reconstruct a structure of the Rev/RRE RNP. Two conformationally locked RREs either alone or in combination with fragment antigen binding (Fabs) generated against the Rev/RRE RNP complex will be used to increase the stability of the complex. It is anticipated that Rev binds to each binding site on the RRE as a dimer utilizing a different dimer interface and crossing angle for each protein-protein or protein-RNA interaction. In aim 2 we will use single particle cryo-EM to reconstruct a structure of the HIV nuclear export complex. In order to increase the affinity of the RNP for Crm1-RanGTP we will use a Rev chimera generated by substituting the native Rev nuclear export sequence (NES) with a high affinity NES. We anticipate that the structure of the Rev/RRE RNP dictates the affinity between Rev and the Crm1 dimer and that the species-specific residues necessary for Crm1 facilitated nuclear export interact directly or indirectly with Rev or the RRE. Understanding the assembly mechanism of the HIV nuclear export complex will provide a prototype for how retroviruses circumvent the host's natural mechanism to retain unspliced mRNAs in the nucleus.